In vitro anti-Plasmodium activity assays.

Inhibition of intraerythrocytic parasite development by benzylmenadione derivatives and control agents was determined by microtiter tests performed according to standard protocols (see below). In vitro antiplasmodial activity is expressed as the 50% inhibitory concentration (IC₅₀) or 90% inhibitory concentration (IC₉₀) for inhibition of parasite development. Activities of plasmodione and chloroquine against the P. falciparum 3D7 and Dd2 strains (as presented in Table 1 and in Table S1 in the supplemental material) were determined in laboratory A by using the SYBR green I assay as described before (8, 29). Briefly, synchronous ring-stage parasites were incubated for 72 h in the presence of decreasing drug concentrations in microtiter plates (final parasitemia, 0.5%; final hematocrit, 1.5%). Each inhibitor was analyzed in 3-fold serial dilutions in duplicate and in at least three independent experiments. Parasite replication was assessed by fluorescent SYBR green staining of parasitic DNA (30) as previously described (29). IC₅₀ and IC₉₀ values were calculated using Prism (GraphPad) [log(inhibitor) versus normalized response − variable slope].

In vitro antimalarial activity against sexual and asexual P. falciparum parasite stages^a

The antiplasmodial activities of the lead compound plasmodione, five benzylmenadione derivatives (1a, 1g, 1h, 1i, and 1j), chloroquine, quinine, monodesethylamodiaquine, and mefloquine against 12 selected P. falciparum strains (as presented in Fig. 2 and in Tables S3 and S4 in the supplemental material) were determined in laboratory C by using the [³H]hypoxanthine incorporation assay (31). Briefly, synchronous parasites at the ring stage (final parasitemia, 0.5%; final hematocrit, 1.5%) were incubated for 48 h in the presence of different drug concentrations in 96-well plates. All strains were synchronized twice with sorbitol before use. Inhibitors were analyzed in 2-fold serial dilutions in duplicate and in 3 to 5 independent repeats. Parasite growth was assessed by adding [³H]hypoxanthine at time zero (31, 32).

In vitro antimalarial activities of plasmodione, chloroquine, and quinine against drug-resistant P. falciparum strains originating from Asia, Africa, or South America. Results are presented as mean IC₅₀s ± 95% confidence intervals for 3 to 5 independent experiments performed with the [³H]hypoxanthine incorporation technique. Strains are ordered according to increasing degree of chloroquine resistance. The chemosusceptibility profiles of the strains and resistance cutoff values are given in Table S2 in the supplemental material. Collective IC₅₀ and IC₉₀ values for all agents are presented in Tables S3 and S4 in the supplemental material. Abbreviations: CQ, chloroquine; QN, quinine; I, intermediate; R, resistant; RS, reduced susceptibility.